The serine protease enterokinase (EK), also known as enteropeptidase, is a heterodimeric glycoprotein present in the duodenal and jejunal mucosa and is involved in the digestion of dietary proteins. Specifically, enterokinase catalyzes the conversion, in the duodenal lumen, of trypsinogen into active trypsin via the cleavage of the acidic propeptide from trypsinogen. The activation of trypsin initiates a cascade of proteolytic reactions leading to the activation of many pancreatic zymogens. (Antonowicz, Ciba Found. Symp., 70: 169-187 (1979); Kitamoto et al., Proc. Natl. Acad. Sci. USA, 91(16): 7588-7592 (1994)). EK is highly specific for the substrate sequence (Asp)4-Lys-Ile on the trypsinogen molecule, where it acts to mediate cleavage of the Lys-Ile bond.
EK isolated from bovine duodenal mucosa exhibits a molecular weight (MW) of 150,000 and a carbohydrate content of 35%. The enzyme is comprised of a heavy chain (MW ˜115,000) and a disulfide-linked light chain (MW˜35,000). (Liepnieks et al., J. Biol. Chem., 254(5): 1677-1683 (1979)). Kitamoto et al., supra, reported that the enterokinase isolated from different organisms exhibits a heavy chain molecular weight variability of from 82-140 kDa and a light chain variability of from 35-62 kDa, depending on the organism. The heavy chain functions to anchor the enzyme in the intestinal brush border membrane and the light chain is the catalytic subunit.
The cloning and functional expression of a cDNA encoding the light chain of bovine enterokinase has been reported. (LaVallie et al., J. Biol. Chem., 268(31): 23311-23317 (1993)). The cDNA sequence codes for a 235 amino acid protein that is highly homologous with a variety of mammalian serine proteases involved in digestion, coagulation and fibrinolysis. The cDNA light chain product migrates at MW 43,000 Da on SDS-PAGE, and exhibits high levels of activity in cleaving the EK-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide.
U.S. Pat. No. 5,665,566 to LaVallie describes the cloning and expression of the enterokinase light chain in CHO cells and Vozza et al., Biotechnology (NY), 14(1): 77-81 (1996) describe the production of rEKL from an expression vector transformed in the methylotrophic yeast Pichia pastoris. 
Lu et al., J. Biol. Chem., 272(50): 31293-31300 (1997) reported that, while the enterokinase light chain, either produced recombinantly or by partial reduction of purified bovine enteropeptidase, had normal activity toward small peptides with the (Asp)4-Lys sequence, the light chain alone had dramatically reduced activity toward trypsinogen compared to the enteropeptidase holoenzyme. Therefore, the recognition of small substrates requires only the light chain, whereas efficient cleavage of trypsinogen may also depend on the presence of the heavy chain. It has been suggested that the improved ability of the light chain alone to cleave the (Asp)4-Lys sequence in fusion proteins with greater efficiency than the holoenzyme may be due to its ability to easily access the pentapeptide depending on its location within the folded fusion protein.
Collins-Racie et al., Biotechnology, 13(9): 982-987 (1995), reported the use of the (Asp)4-Lys pentapeptide substrate in a fusion protein as an autocatalytic substrate for the production of recombinant light chain enterokinase (rEKL). Essentially, rEKL cDNA was fused in frame to the C-terminus of the coding sequence for E. coli DsbA protein, which directs secretion to the E. coli periplasmic space. These two domains were joined by the (Asp)4-Lys linker/cleavage sequence fused immediately upstream to the N-terminus of the mature rEKL domain. Collins-Racie et al. recovered a soluble DsbA/rEKL fusion protein from cells expressing the gene fusion construct. Following partial purification of the fusion protein, active rEKL was recovered subsequent to autocatalysis of the (Asp)4-Lys pentapeptide.
Wang et al., Biol. Chem. Hoppe Seyler, 376(11): 681-684 (1995) describe the production of enzymatically active recombinant human chymase (rHC), a proteinase present in mast cells, by a method involving proteolytic activation from a ubiquitin fusion protein containing the enterokinase cleavage site in place of the native chymase propeptide. Wang et al. transformed E. coli with an expression vector comprising the coding sequence for ubiquitin linked to the enterokinase cleavage sequence linked to the chymase gene. The fusion protein was expressed and analyzed for enterokinase-mediated activation of chymase from the refolded fusion protein. At the highest concentration of enterokinase, approximately 2.5% of the folded fusion protein was converted into enzymatically active rHC, as evidenced in comparative studies with human chymase. From these analyses, Wang et al. concluded that the use of the enterokinase cleavage site in place of the native propeptide for activation purposes, demonstrates that the presence of the native propeptide is not essential for the folding and activation of HC expressed in recombinant systems.
Light et al., Anal. Biochem., 106: 199-206 (1980) investigated the specificity of the enterokinase holoenzyme purified to homogeneity from bovine intestinal mucosa through incubation of the enzyme with various proteins of known sequence followed by an analysis of the resulting fragments on SDS-PAGE. Analysis of the resulting protein fragments indicated that either lysine or arginine can occupy the amino acid position immediately upstream (towards the amino-terminus) of the cleaved peptide bond (the P1 position), an acidic amino acid must occur immediately upstream of this lysine or arginine (the P2 position) and hydrolysis was increased when an acidic amino acid occurred at the 2nd and 3rd amino acids upstream from the cleaved peptide bond (the P2 and P3 positions).
Additionally, Light and Janska, Trends Biochem. Sci., 14(3) 110-112 (1989), reported studies showing that lysyl, arginyl, or the cysteinyl derivative, S-aminoethyl cysteine, could be substituted for the basic lysine residue and that aspartyl, glutamyl, or S-carboxymethyl cysteine could be substituted for the basic arginine residues. Additionally, they reported that asparagine at the 3rd amino acid position upstream from the cleaved peptide bond (known as the “scissile bond”) slowed hydrolysis by enterokinase and that changes at the 4th and 5th upstream positions showed greater variability but also slowed the rate of hydrolysis.
Presently, while current investigations into the advantages of utilizing the highly specific (Asp)4-Lys enterokinase recognition sequence for various chemical and biological applications are promising, these potential applications are hindered by the enzyme/substrate kinetics which act to limit specificity and rate of hydrolysis. Therefore, since enterokinase, both natural and recombinant, is readily available in commercial quantities, it would be advantageous to identify additional enterokinase cleavage sequences that exhibit an even higher specificity as well as a higher rate of hydrolysis than currently observed with the (Asp)4-Lys pentapeptide recognition sequence.
In particular, the discovery of new peptides that are cleaved rapidly and specifically by enterokinase would find beneficial use in the field of large scale protein purification.